The goal of this project is to develop a highly potent antitoxin for the treatment and prevention of botulism caused by type F botulinum neurotoxin (BoNT/F). The antitoxin will consist of a combination of three or four human or humanized monoclonal antibodies (mAbs) which together bind to and neutralize the seven BoNT/F sub-serotypes. BoNTs are one of the six highest-risk threat agents for bioterrorism, due to their extreme potency and lethality, ease of production, and need for prolonged intensive care. The mainstay of treatment is equine antitoxin, made by hyperimmunizing horses. Equine antitoxin has a reduced potency for sub-serotypes, a significant incidence of allergic reactions, a short serum half-life leading to reintoxication, is challenging to administer, and cannot be given prophylactically. We have generated combinations of three mAbs that bind all sub-serotypes tested of BoNT/A, B, or E and result in highly potent BoNT neutralization in vivo. Combining three mAbs binding non-overlapping epitopes leads to highly potent BoNT neutralization due to rapid clearance of BoNT from the circulation. Based on the potency, HHS has awarded contracts to XOMA (US) LLC, Berkeley, CA to develop these mAb combinations for clinical use. The BoNT/A mAb combination has completed dosing in a Phase 1 clinical trial. The BoNT/B and BoNT/E mAb combinations will enter clinical trials by second quarter 2014. BoNT/F is particularly challenging, as there are seven known sub-serotypes, which differ from each other by up to 33% at the amino acid level. Sub-serotype sequence variation results in reduced potency of equine antitoxin and makes finding mAbs that bind and neutralize all sub-serotypes challenging. Under NIAID U01 funding, we have developed a panel of lead mAbs binding all or many of the BoNT/F sub- serotypes. We have also shown that when three of these mAbs bind with high affinity to a BoNT/F sub- serotype, potent in vivo neutralization occurs. For this projec, we hypothesize that these lead mAbs can be engineered to improve affinity and cross reactivity for the BoNT/F sub-serotypes and then developed into a three or four mAb combination that potently neutralizes all BoNT/F sub-serotypes. We will accomplish this by completing the following specific aims. Aim 1. Evaluate and optimize individual lead mAb characteristics to identify lead combinations of three or four mAbs that bind and neutralize the seven reported BoNT/F sub-serotypes. Aim 2. Generate non-toxic BoNT/F domains and assays specific for each individual mAb. Aim 3. Develop stable cell lines that express mAbs. Aim 4. Conduct initial process investigations using two-step chromatography and achieve 90% pure antibody. At the completion of this project, we will have a preclinical lead candidate BoNT/F antitoxin consisting of three or four human or humanized mAbs that have acceptable human tissue cross reactivity, bind and neutralize all seven BoNT/F sub-serotypes, and have stable cell lines that express these antibodies. The stable CHO cell lines produced in this project can be rapidly developed in the future as master and working cell banks for GMP manufacture and clinical development.